In the recent past immense research has been dedicated to the discovery and understanding of the structure and functions of enzymes and bio-molecules associated with various diseases. One such important class of enzymes that has been the subject of extensive research is dihydroorotate dehydrogenase (DHODH).
DHODH is an enzyme that catalyzes the fourth step in the de novo biosynthesis of pyrimidine. It converts dihydroorotate (DHO) to orotate (ORO). Human DHODH is a ubiquitous flavine mononucleotide (FMN) moiety flavoprotein. In bacteria (gene pyrD), it is located on the inner side of the cytosolic membrane. In some yeasts, such as in Saccharomyces cerevisiae (gene URA1), it is a cytosolic protein while in other eukaryotes it is found in the mitochondria (see Proc. Natl. Acad. Sci. U.S.A., 89 (19), 8966-8970).
DHODH has been classified as a family of class I or class II proteins on the basis of the cofactor. Human DHODH belongs to the family class 2 that utilizes flavine as a redox cofactor, unlike the bacterial family class 1 protein that uses fumarate or NAD+ instead. In the cell the mammalian protein is anchored at the inner mitochondrial leaflet. There, DHODH catalyzes the conversion of DHO to ORO, which represents the rate limiting step in the de novo pyrimidine biosynthesis. (see McLean et al., Biochemistry 2001, 40, 2194-2200). Kinetic studies indicate a sequential ping-pong mechanism for the conversion of DHO to ORO (see Knecht et al., Chem. Biol. Interact. 2000, 124, 61-76). The first half-reaction comprises the reduction of DHO to ORO. Electrons are transferred to the FMN which becomes oxidized to dihydroflavin mononucleotide (FMNH2). After dissociation of ORO from the enzyme, FMNH2 is regenerated by a ubiquinone molecule, which is recruited from the inner mitochondrial membrane. Kinetic and structural studies revealed two distinct binding sites for DHO/ORO and ubiquinone, respectively.
Human DHODH is composed of two domains, a large C-terminal domain (Met78-Arg396) and a smaller N-terminal domain (Met30-Leu68), connected by an extended loop. The large C-terminal domain can be best described as an α/β-barrel fold with a central barrel of eight parallel β strands surrounded by eight α helices. The redox site, formed by the substrate binding pocket and the site that binds the cofactor FMN, is located on this large C-terminal domain. The small N-terminal domain, on the other hand, consists of two α helices (labeled α1 and α2), both connected by a short loop. This small N-terminal domain harbors the binding site for the cofactor ubiquinone. The helices α1 and α2 span a slot of about 10×20 Å2 in the so-called hydrophobic patch, with the short α1-α2 loop at the narrow end of that slot. The slot forms the entrance to a tunnel that ends at the FMN cavity nearby the α1-α2 loop. This tunnel narrows toward the proximal redox site and ends with several charged or polar side chains (Gln47, Tyr356, Thr360, and Arg136). Structural clues, as discussed above, along with kinetic studies suggest that ubiquinone, which can easily diffuse into the mitochondrial inner membrane, uses this tunnel to approach the FMN cofactor for the redox reaction (see Baumgartner et al., J. Med. Chem. 2006, 49, 1239-1247).
A study disclosed in The Journal of Biological Chemistry 2005, 280(23), 21847-21853; formally demonstrates the possibility to identify potent inhibitors of P. falciparum DHODH that do not inhibit the human enzyme. Comparison of the human DHODH crystal structures with the malaria DHODH amino acid sequence further suggests there are opportunities for species-specific inhibitor binding.
In the body, DHODH catalyzes the synthesis of pyrimidines, which are necessary for cell growth. An inhibition of DHODH inhibits the growth of (pathologically) fast proliferating cells, whereas cells which grow at normal speed may obtain their required pyrimidine bases from the normal metabolic cycle. The most important types of cells for the immune response, the lymphocytes, use exclusively the synthesis of pyrimidines for their growth and react particularly sensitively to DHODH inhibition.
DHODH inhibition results in decreased cellular levels of ribonucleotide uridine monophosphate (rUMP), thus arresting proliferating cells in the Gl phase of the cell cycle. The inhibition of de novo pyrimidine nucleotide synthesis is of great interest in view of the observations that lymphocytes seem not to be able to undergo clonal expansion when this pathway is blocked. Substances that inhibit the growth of lymphocytes are important medicaments for the treatment of auto-immune diseases.
During homeostatic proliferation, the salvage pathway which is independent of DHODH seems sufficient for the cellular supply with pyrimidine bases. Only, cells with a high turnover and particularly T and B lymphocytes need the de novo pathway to proliferate. In these cells, DHODH inhibition stops the cell cycle progression suppressing DNA synthesis and consequently cell proliferation (see Breedveld et al., Ann Rheum Dis 2000).
Therefore, inhibitors of DHODH show beneficial immunosuppressant and antiproliferative effects in human diseases characterized by abnormal and uncontrollable cell proliferation causing chronic inflammation and tissue destruction. The human enzyme dihydroorotate dehydrogenase (DHODH) represents a well-characterized target for small molecular weight Disease Modifying Antirheumatic Drugs (DMARDs).
A list of known DHODH inhibitors includes Leflunomide, Teriflunomide, Brequinar (NSC 368390) (Cancer Research 1992, 52, 3521-3527), Dichloroallyl lawsone (The Journal of Biological Chemistry 1986, 261(32), 14891-14895), Maritimus (FK 778) (Drugs of the Future 2002, 27(8), 733-739) and Redoxal (The Journal of Biological Chemistry 2002, 277(44), 41827-41834),

Leflunomide, teriflunomide, and brequinar have been studied significantly.
In general, inhibitors of DHODH show beneficial immunosuppressive and antiproliferative activities, most pronounced on T-cells (see Fairbanks et al., J. Biol. Chem. 1995, 270, 29682-29689). Brequinar and leflunomide are two examples of small molecular weight inhibitors of DHODH that had been in clinical development. The latter is used in the treatment of rheumatoid arthritis refractive to methotrexate (see Rozman J. Rheumatol Suppl. 1998, 53, 27-31; Pally et al., Toxicology 1998, 127, 207-222). Clinical application of both molecules suffers from various side effects. On the basis of very good efficacy in animal models, brequinar was originally developed for the therapy of organ transplant rejection but was switched to cancer as a secondary indication. The compound failed in the clinic due to its narrow therapeutic window. Oral administration of brequinar and some of its analogues resulted in toxic effects, including leukocytopenia and thrombocytopenia, when given in combination with cyclosporine. The application of leflunomide might be flawed by its long half-life time of approximately 2 weeks which represents a serious obstacle in patients that have developed side effects (see Fox et al. J. Rheumatol Suppl. 1998, 53, 20-26; Alldred et al., Expert Opin. Pharmacother. 2001, 2, 125-137).
In addition to abolish lymphocyte proliferation, inhibitors of DHODH (e.g., teriflunomide, maritimus (FK778) and brequinar) have an anti-inflammatory action by inhibition of cytokine production and nuclear factor (NF)-kB-signalling, monocyte migration and increased production of transforming growth factor beta-1 and induce a shift from T helper cell type 1 (TM) to type 2 (Th2) subpopulation differentiation (Manna et al., J. Immunol. 2000; Dimitrova et al., J. Immunol. 2002). Furthermore, the osteoclast differentiation mediated by Receptor Activator for Nuclear Factor k B Ligand (RANKL) decreased by DHODH inhibition (Urushibara et al., Arthrititis Rheum 2004). In co-crystallisation experiments with two inhibitors of DHODH that reached clinical trials, brequinar (Dexter et al., Cancer Res. 1985) and teriflunomide (A77-1726), were both found to bind in a common site, that is also believed to be the binding site of the cofactor ubiquinone (Liu et al., Struc. Fold. Des. 2000).
Leflunomide sold under the trade name Arava (EP 0 780 128, WO 97/34600), was the first DHODH inhibitor that reached the market place. Leflunomide is the prodrug of teriflunomide, which is the active metabolite inhibiting human DHODH with a moderate potency (Fox et al., J. Rheumatol. Suppl. 1998).
Leflunomide is a DMARD from Aventis, which was approved by the FDA for the treatment of rheumatoid arthritis in 1998 and by the EMEA for the treatment of psoriatic arthritis in 2004. Currently leflunomide is under active development for the treatment of systemic lupus erythematosus, Wegener's granulomatosis (Metzler et al., Rheumatology 2004, 43(3), 315-320) and HIV infection. Moreover, teriflunomide, its active metabolite is efficacious in multiple sclerosis and is currently in Phase III clinical trials (O'Connor et al., Neurology 2006).
Other data are emerging in other closely related diseases such as ankylosing spondilitis (Haibel et al., Ann. Rheum. Dis. 2005), polyarticular juvenile idiopathic arthritis (Silverman et al., Arthritis Rheum. 2005) and Sarcoidosis (Baughman et al., Sarcoidosis Vase. Diffuse Lung Dis. 2004). Furthermore, leflunomide and FK778 have shown antiviral activity against cytomegalovirus. Leflunomide is currently indicated as second-line therapy for cytomegalovirus disease after organ transplantation (John et al., Transplantation 2004). In addition leflunomide reduces HIV replication by about 75% at a concentration that can be obtained with conventional dosing (Schlapfer et al., AIDS 2003).
DHODH inhibitors under investigation at various stages of clinical trials are 4SC-101 (Phase-II) from 4SC AG; LAS-186323 (Phase-I) from Almirall Laboratories SA and ABR-224050, ABR-222417, & ABR-214658 (Preclinical) from Active Biotech AB. The exact structures of all these molecules have not yet been disclosed.
Various DHODH inhibitors have been disclosed for the treatment or prevention of autoimmune diseases, immune and inflammatory diseases, destructive bone disorders, malignant neoplastic diseases, angiogenic-related disorders, viral diseases, and infectious diseases. See for example WO2009137081; WO2009133379; WO 2009021696; WO2009082691; WO2009029473; WO2009153043; US2009209557; US2009 062318; US2009082374; WO2008097180; WO2008077639; US2008027079; US2007 299114; US2007027193; US2007224672; WO2007149211; JP2007015952; WO2006 044741; WO2006001961; WO2006051937; WO2006038606; WO2006022442; US2006 199856; WO2005075410; U.S. Pat. No. 7,074,831; WO2004056797; U.S. Pat. No. 7,247,736; WO2004056747; WO 2004056746; JP2004099586; WO2003097574; WO2003030905; WO2003006425; WO2003 006424; US2003203951; WO2002080897; U.S. Pat. No. 7,176,241; U.S. Pat. No. 7,423,057; WO2001024785; U.S. Pat. No. 6,841,561; WO9945926; WO9938846; WO9941239; EP767167 and U.S. Pat. No. 5,976,848.
For additional reviews and literature regarding DHODH inhibitors see Bio & Med. Chem. Letters, 20(6), 2010, Pages 1981-1984; J. Med. Chem. 2009, 52, 2683-2693; J. Med. Chem. 2008, 51 (12), 3649-3653. All of these patents, patent applications, and literature disclosures are incorporated herein as reference in their entirety for all purposes.
Despite the progress made in the area of DHODH inhibition in human diseases, challenges remain in terms of the side effects and desired clinical benefits from small molecule inhibitors. Accordingly, there still remains an unmet and dire need for small molecule DHODH inhibitors for the treatment and/or amelioration of diseases and disorders known to be associated with DHODH.